Anthrax disease results from a complex series of interactions between the invading bacterium, Bacillus anthracis, and the mammalian host. For inhalation anthrax, infection begins with entry of spores into the lung. Alveolar macrophages phagocytose the spores and transport them to lymph nodes of the mediastinum. Ultimately the metabolically active form of the bacterium disseminates to the blood and other body tissues, reaching concentrations up to 10(8) CFU per ml and secreting the anthrax toxin proteins. In recent years, research emphases have focused on toxin protein structure and function. However, anthrax disease, whether acquired naturally or as the result of intentional dissemination of spores, results from infection with B. anthracis, not simply acquisition of toxin. Despite the importance of human infection with B. anthracis, there is an almost complete lack of knowledge of fundamental cellular and molecular mechanisms by which the bacterium interacts with its host. Results of studies proposed here will fill this critical gap in knowledge and reveal bacterial and host targets for generation of new therapeutics for anthrax. We will use an in vitro macrophage model and in vivo murine model to identify pathogen and host targets important for multiple early steps in infection. The importance of pathogen and host factors during early infection will be assessed in both models by modulating expression of candidate B. anthracis and macrophage targets. In Aim 1 we will identify and characterize Bacillus anthracis and macrophage molecular targets important for multiple steps of early infection. We will establish a detailed model of B. anthracis-macrophage interactions. A major part of this work will be to characterize the modulation of both bacterial and macrophage gene expression as a result of B. anthracis-macrophage interactions, using transcriptional profiling and proteome analyses. In Aim 2 we will investigate B. anthracis development in a mouse nasal installation model for anthrax, focusing on the pulmonary response. We will test B. anthracis mutants for attenuation of pathogenesis in the model. B. anthracis germination, survival, and persistence in the lung will be correlated with lung histopathology and immune response. We will track development of B. anthracis in the whole animal using chemoluminescence-based in vivo imaging technology. Using these assays, we will establish the spatial and temporal development of a fully virulent B. anthracis strain and isogenic mutants deleted for genes encoding therapeutic candidates. Our long-term objective is to generate new therapeutics to block interactions of B. anthracis spores with alveolar macrophages. The most powerful strategy will probably employ a cocktail of inhibitors targeting multiple steps in the infectious process. Bacterial and macrophage targets shown experimentally to be important for B. anthracis-macrophage interactions will be immediately forwarded to RCE core-facilities to be expressed recombinantly and crystallized for high-resolution structural analysis. The structural data will be used for structural-based identification of lead-inhibitor templates.